丙型肝炎病毒疫苗研究进展

近年来,在人体内及黑猩猩试验模型中发现HCV天然免疫与特异性免疫能自发清除病毒,表明发展部分有效的针对不同型的HCV疫苗是可能的,这对慢性丙型肝炎的防治有重大意义。此文综述了HCV感染相关免疫保护机制、疫苗研制情况及今后发展方向。     

Therapeutic vaccines against hepatitis C virus

Hepatitis C virus (HCV) is a blood-borne pathogen which has chronically infected about 130–210 million people worldwide. Current standard-of-care (SoC) therapy is an inadequate and expensive treatment with more side effects. Two direct-acting antiviral agents (DAAs) (telaprevir and boceprevir) in combination with SoC therapy have been used in patients infected with HCV genotype 1. Although these drugs result in a shortening of therapy, they also have additional side effects and are expensive. In their stead, several second-generation DAAs are being investigated. What important is that all-oral, interferon (IFN)- and ribavirin-free regimens for the treatment of HCV-infected patients are now being investigated, and will be applied in the next year. Preventive measures against HCV, including vaccine development, are also now in progress. However, no therapeutic vaccine against HCV has been produced to date. An effective vaccine should induce robust and broadly cross-reactive CD4⁺, CD8⁺T-cell and neutralising antibody (NAb) responses. Current data indicate that vaccines can usually not completely prevent HCV infection but rather prevent the progression of HCV infection to chronic and persistent infection, which may be a realistic goal. This review discusses the important roles of NAbs and CD8⁺T-cells in the development of therapeutic vaccines, and summarizes some important epitopes of HCV recognized by CD8⁺T-cells and some prospective therapeutic vaccine approaches.     

联合检测丙肝病毒核心抗原和抗体在丙型肝炎早期诊断中的价值

目的:通过检测丙型肝炎抗体(HCV-Ab),同时对其进行丙型肝炎病毒核心抗原(HCV-cAg)和丙肝病毒RNA(HCV-RNA)两项指标进行检测,探讨HCV-cAg检测在丙型肝炎早期诊断中的意义。方法:对104例HCV-Ab阳性标本及108例高危人群但HCV-Ab阴性标本(包括血液透析患者55例、医务人员20例和丙型肝炎病人的家庭密切接触者33例)及健康对照组同时进行HCV-cAg和HCV-RNA的检测。结果:104例HCV-Ab阳性组中,HCV-cAg阳性25例,HCV-RNA阳性27例;108例HCV-Ab阴性组中,HCV-cAg阳性2例,HCV-RNA阳性1例;健康对照组全阴性。结论:HCV-cAg和HCV-RNA在丙型肝炎早期诊断上无显著性差异,但HCV-cAg检测在临床的应用前景上具有更大的优势,故可在临床推广HCV-Ab和HCV-cAg联合检测,有条件者可联合检测HCV-Ab、HCV-cAg和HCV-RNA。     

Indications of immune protection from hepatitis C infection

Hepatitis C affects many millions of people worldwide and is at very high prevalence among people who inject drugs. In our study of hepatitis C virus (HCV) in the social networks of injecting drug users (IDUs), five IDUs with injecting careers of 9 years or more were HCV antibody and RNA negative. All injected frequently with HCV RNA-positive IDUs, and two had recently injected with the syringe of an RNA-positive IDU. Our data suggest the existence of immune protection from HCV infection.     

Sexual transmission of hepatitis C virus infection

The role of sexual transmission in the diffusion of HCV infection, was studied through the seroprevalence of anti-HCV antibodies in the heterosexual habitual partners of 83 anti-HCV positive subjects. The index cases were represented by 10 dialysed subjects, 31 patients with chronic liver disease and 42 healthy carriers. Seroprevalence of anti-HCV positivity reported in partners was 8.43%, with a higher rate in cohabitants of patients with chronic liver disease (16.12% vs 4.76% of carriers); no case was found among partners of dialysed subjects. Laboratory and ultrasonograph signs of chronic hepatitis were reported in 3 cases (3.61%). Control on 70% of the cohabitants' relatives, was negative for HCV infections. These data suggest a possible sexual transmission of HCV infection, even if its prevalence resulted modest, undoubtedly lower than in other disease sexually transmitted.     

Antibodies to hepatitis C virus in blood donors

Recently a recombinant polypeptide of hepatitis C virus (HCV) has been developed by the Chiron Corporation in California. This antigen has been used to develop an ELISA test (Ortho Diagnostic Systems) for serum anti-HCV antibodies. Preliminary data have shown that this virus is the major cause of NANB hepatitis in the world. We examined differences in anti-HCV prevalence among subgroups of blood donors (total sera examined 639) classified for past or present exposure to HBV or not, and for ALT levels. The anti-HCV prevalence found in regular blood donors with normal ALT levels and no antibody to HBcAg was 1.2%. No significant difference in the anti-HCV prevalence was found among other subgroups of blood donors except that a higher prevalence (10%) was found in a group with both elevated ALT and HBV markers.These preliminary findings suggest that the policy of blood supply should take into account the advent of HCV antibody test.     

Murine neutralizing antibody response and toxicity to synthetic peptides derived from E1 and E2 proteins of hepatitis C virus

Introduction

The highest estimated prevalence of HCV infection has been reported in Egypt, nearly 12% mostly type 4. Currently, a commercial vaccine to protect this high risk population as well as global HCV infected patients is not available.

Objectives

In the present study, we aim at: (1) examining the viral binding capacities of purified monospecific polyclonal murine antibodies raised against genetically conserved viral protein sequences, i.e. synthetic peptides derived from those sequences located within envelope proteins and (2) assessment of immunogenic properties and safety parameters of those peptides individually and in a vaccine format in mice.

Methods

Purified IgG Abs from immunized mice were used in immunocapture RT-PCR experiments to test viral neutralization by Abs raised against each of 4 peptides termed p35 (E1), p36 (E2), p37 (E2) and p38 (E2). Swiss mice were immunized with each of the 3 peptides (p35, p37 and p38) which generated neutralizing antibodies in immunocapture experiments. Antibody responses to corresponding peptides were determined using different routes of administration, different adjuvants, different doses and at different time points post-injection. To explore the dose range for future pharmacological studies, three doses namely 50 ng, 10 μg and 50 μg/25 gm mouse body weight were tested for biochemical and histopathological changes in several organs.

Results

Murine Abs against p35, p37 and p38 but not p36 showed HCV neutralization in immunocapture experiments. Subcutaneous injection of peptides elicited higher responses than i.m. and i.p. Immunization with Multiple Antigenic Peptide (MAP) form or coupled to Al PO4 elicited the highest Ab responses. Peptide doses of 50 ng/25 gm body weight or less were effective and safe, however dose assessment still requires further study. Histopathological changes were observed in animals that received doses ∼1000 times higher than the potential therapeutic dose.

Conclusion

Exploration of humoral immunogenicity, neutralization capacity and safety suggested that the peptides presented herein are candidate vaccine components for further preclinical assessment.     

Major decline of hepatitis C virus incidence rate over two decades in a cohort of drug users

Injecting drug users (DU) are at high risk for hepatitis C virus (HCV) and HIV infections. To examine the prevalence and incidence of these infections over a 20-year period (1985–2005), the authors evaluated 1276 DU from the Amsterdam Cohort Studies who had been tested prospectively for HIV infection and retrospectively for HCV infection. To compare HCV and HIV incidences, a smooth trend was assumed for both curves over calendar time. Risk factors for HCV seroconversion were determined using Poisson regression. Among ever-injecting DU, the prevalence of HCV antibodies was 84.5% at study entry, and 30.9% were co-infected with HIV. Their yearly HCV incidence dropped from 27.5/100 person years (PY) in the 1980s to 2/100 PY in recent years. In multivariate analyses, ever-injecting DU who currently injected and borrowed needles were at increased risk of HCV seroconversion (incidence rate ratio 29.9, 95% CI 12.6, 70.9) compared to ever-injecting DU who did not currently inject. The risk of HCV seroconversion decreased over calendar time. The HCV incidence in ever-injecting DU was on average 4.4 times the HIV incidence, a pattern seen over the entire study period. The simultaneous decline of both HCV and HIV incidence probably results from reduced risk behavior at the population level. Charlotte H.S.B. van den Berg and ColetteSmit contributed equally to this paper     

A novel linear neutralizing epitope of hepatitis E virus

Hepatitis E virus (HEV) is a serious public health problem that causes acute hepatitis in humans and is primarily transmitted through fecal and oral routes. The major anti-HEV antibody responses are against conformational epitopes located in a.a. 459–606 of HEV pORF2. All reported neutralization epitopes are present on the dimer domain constructed by this peptide. While looking for a neutralizing monoclonal antibody (MAb)-recognized linear epitope, we found a novel neutralizing linear epitope (L2) located in a.a. 423–437 of pORF2. Moreover, epitope L2 is proved non-immunodominant in the HEV-infection process. Using the hepatitis B virus core protein (HBc) as a carrier to display this novel linear epitope, we show herein that this epitope could induce a neutralizing antibody response against HEV in mice and could protect rhesus monkeys from HEV infection. Collectively, our results showed a novel non-immunodominant linear neutralizing epitope of hepatitis E virus, which provided additional insight of HEV vaccine.     

丙型肝炎免疫发病机制的研究进展

丙型肝炎病毒(HCV)是输血后肝炎的主要致病因子,给人类的健康造成了极大的危害。由于HCV基因的高变性,容易逃避机体免疫系统的清除;HCV感染免疫系统细胞,造成机体对HCV感染的免疫应答异常,并引起组织的损伤,导致肝细胞的慢性持续性感染,并可进一步发展成肝硬化,甚至肝细胞肝癌。文中对近年来HCV在免疫致病机制方面的研究进展进行了综述。     

宁波地区丙型肝炎病毒基因分型检测

目的:通过检测丙型肝炎病毒基因分型,探讨宁波地区HCV感染基因型流行株特征。方法:本文共收集231例HCV感染者的血清,采用基因芯片技术进行HCV基因分型。结果:231例丙型肝炎病毒基因分型1b型162例,占70.12%;2a型25例,占10.82%;1b+2a混合型15例,占6.49%;并有少数的3b3、a6、型和其它混合型,均占<5%。结论:宁波地区HCV感染基因型以lb型为主、其次为2a型。基因芯片法检测丙型肝炎病毒基因分型,具有微量、多位点、准确性好、灵敏度高、特异性强等特点。     

体外转录法制备地高辛标记HCV核心区RNA探针及其初步应用

目的制备地高辛标记的HCV核心区RNA探针,用于筛选与丙型肝炎病毒(HCV)RNA核心区结合的细胞蛋白质分子。方法采用RT-PCR方法,从HCV感染者血清中扩增HCV C区cDNA序列,将所得序列克隆入载体PinpointT-MT Vector和pGEM-7EF( )Vector,进行PCR、酶切和测序鉴定。以HCV-pGEM-7EF( )中的HCV核心区cDNA序列作为体外转录RNA的模板,经T7 RNA聚合酶转录得到地高辛标记的HCV核心区RNA探针。将地高辛标记的探针用于紫外交联实验,筛选HepG2细胞中可与HCV RNA结合的蛋白质分子。结果所得HCV核心区cDNA序列为503 bp。地高辛标记的HCV核心区探针初步筛选到两个与HCV RNA结合的蛋白。结论地高辛标记的HCV核心区RNA探针具有较高的特异性和敏感性,可用于筛选与HCV RNA结合的细胞蛋白质分子。     

Assessing the feasibility of hepatitis C virus vaccine trials: Results from the Hepatitis C Incidence and Transmission Study-community (HITS-c) vaccine preparedness study

Efficacy trials of preventive hepatitis C virus (HCV) vaccine candidates raise challenging scientific and ethical issues. Based on data from the first 3 years of a community-based prospective observational study – the Hepatitis C Incidence and Transmission Study-community (HITS-c) – this paper examines the feasibility of conducting trials of candidate HCV vaccines with people who inject drugs (PWID) in Sydney, Australia. Of the 166 PWID confirmed HCV antibody negative and eligible for enrolment, 156 (94%) completed baseline procedures. Retention was high, with 89% of participants retained at 48 weeks and 76% of participants completing at least 75% of study visits within 2 weeks of schedule. The rate of primary HCV infection was 7.9/100 py (95% CI 4.9, 12.7). Of the 17 incident cases, 16 completed at least one follow-up assessment and 12 (75%) had evidence of chronic viraemia with progression to chronic HCV infection estimated to be 6/100 py. Power calculations suggest a chronic HCV infection rate of at least 12/100 py (primary HCV infection rate 16/100 py) will be required for stand-alone trials of highly efficacious candidates designed to prevent chronic infection. However, elevated primary HCV infection was observed among participants not receiving opioid substitution therapy who reported heroin as the main drug injected (26.9/100 py, 95% CI 14.5, 50.0) and those who reported unstable housing (23.5/100 py, 95% CI 7.6, 72.8), daily or more frequent injecting (22.7/100 py, 95% CI 12.2, 42.2) and receptive syringe sharing (23.6/100 py, 95% CI 9.8, 56.7) in the 6 months prior to baseline. These data suggest that it is possible to recruit and retain at-risk PWID who adhere to study protocols and that modification of eligibility criteria may identify populations with sufficiently high HCV incidence. Results support the feasibility of large multi-centre HCV vaccine trials, including in the Australian setting.     

丙型肝炎病毒全基因组感染细胞模型研究

HCV是导致慢性肝炎、肝纤维化、肝细胞癌的重要致病因子,建立HCV全基因组感染细胞模型对于研究HCV致病机制,研究抗HCV药物具有重要意义,此文试就该领域内目前研究成果进行综述。     

静脉注射毒品人群中HIV、HBV和HCV感染的现况研究

目的了解静脉注射毒品人群中人类免疫缺陷病毒（HIV）、乙型肝炎病毒（HBV）和丙型肝炎病毒（HCV）的感染情况。方法从四川、湖南、广西和新疆等地静脉注射毒品人群中采集血液样本2025份，应用酶联免疫试剂盒检测抗-HIV、抗-HCV抗体和HBsAg。结果红静脉沣射毒品人群中，抗-HIV、抗-HCV及HBsAg的阳性率分别为14．7％～30．4％、60．7％～85．5％和6．6％～22．4％，其HIV／HBV、HIV/HCV、HCV／HBV和HIV／HCV／HBV合并感染率分别为0％～0．4%、11．6％～27．2％、2．3％～14．3％和1．6％～4．8％。结论静脉注射毒品人群中HIV、HBV和HCV的感染率均高于正常人群，其中HIV与HCV合并感染率最高。     

江苏省宜兴地区丙型肝炎病毒基因分型研究

目的了解江苏省宜兴地区丙型肝炎病毒（HCV）基因型分布特征和病毒变异情况。方法对宜兴市人民医院收治的158例HCV抗体阳性患者血清进行HCV RNA检测，对阳性标本进行Simmonds分型，采用5’非编码区（5’NCR）1、2、3、1b型特异性引物进行PCR扩增，并对1b型和2型各1株的PCR产物测序验证。分析不同性别、临床类型的丙型肝炎患者基因型分布差异。结果158份血清中，有95份为HCV RNA阳性，其中1b型80份（84．2％），2型5份（5．3％），1b／2型的混合感染5份（5．3％），不能分型的5份（5．3％），1b型的序列与GenBank J-4株的同源性为99．2％，2型与GenBank J-6株的同源性为97．7％。男性和女性在基因型总体分布上存在差异（P〈0．05），并且在单一型感染和混合型感染的分布上差异亦有统计学意义，不同临床类型的丙型肝炎中HCV基因型分布未显示差异。结论江苏省宜兴地区HCV以1b型为主，男女患者HCV基因型分布差异有统计学意义，而不同临床类型的HCV基因型分布无显著差异，基本反映了本地区HCV感染的特点。     

The immune response of rhesus macaques to novel vaccines comprising hepatitis B virus S,PreS1, and Core antigens

Therapeutic vaccines represent a unique approach to hepatitis B virus (HBV) treatment and have the potential to induce long-term control of infection. This study explored the immune responses of rhesus macaques to novel vaccines comprising the S, PreS1, and Core antigens of the HBV that showed promise as prophylactic and therapeutic approaches in a mouse model. The tested vaccines included two DNA vaccines (pVRC-SS1, pVRC-CS1), an HBV particle subunit (HBSS1) vaccine and the recombinant vaccinia virus- (RVJ-) based vaccines (RVJSS1 and RVJCS1) in which SS1 containing S (1–223 aa) and PreS1 (21–47 aa), CS1 containing Core (1–144 aa) and PreS1 (1–42 aa). The humoral immunity and cell-mediated immunity (CMI) induced by vaccines comprising the S, PreS1, and Core antigens of HBV were investigated in a longitudinal study that continued up to 98 weeks after the first vaccination. In rhesus macaques, anti-PreS1 antibody was induced more rapidly than anti-S or anti-Core antibody after DNA vaccination. The antibody and cell-mediated immune responses against S, PreS1, and C were significantly enhanced in macaques boosted with RVJSS1 and RVJCS1, whereas the cell-mediated response to C was most robust and durable. The immune response to S, PreS1, and C was restored by HBSS1 boosting and detected in macaques until weeks 74 and 98 after the first vaccination. Additionally, robust neutralizing activity was detected at week 52. In conclusion, novel HBV vaccine candidates, especially those used for therapeutic applications should incorporate the PreS1 and Core antigens.     

海口地区丙型肝炎病毒基因分型研究

目的 为制定治疗方案提供参考,了解海口地区丙型肝炎病毒基因分型的特征.方法 选择2010年1月～2011年1月某院经临床检测HCV-RNA阳性的72例患者血清,回顾分析HCV测序分型结果、套式PCR结果,对照分析不同HCV-RNA阳性人群HCV基因分型及不同基因型HCV-RNA定量检测值.结果 本组共72份血清标本,基因型构成比次序依次为:1b型构成比为62.5％ (45/72);2a型构成比为29.2％ (21/72); 3a型构成比为8.3％ (6/72).不同HCV-RNA阳性人群HCV基因分型结果对照显示,差异无统计学意义(P＞0.05).1b型HCV-RNA定量检测值显著高于2a型和3a型(P＜0.05),2a型HCV-RNA定量检测值显著高于3a型(P＜0.05).结论 海口地区丙型肝炎病毒基因分型和国内多数地区相同,以1b型为主,2a型次之,同时基因分型也有助于了解患者体内病毒复制情况,为治疗提供参考.     

丙型肝炎病毒感染与糖尿病

近年来,有很多流行病学调查证实了HCV感染与糖尿病的关联,这种关联在年龄大、体重超标的患者身上表现更为明显。HCV编码的蛋白,能干扰胰岛素的信号转导,这就解释了在发生肝纤维化之前,HCV感染者出现胰岛素抵抗的原因,而胰岛素抵抗也促进了肝纤维化的进程。     

浙江口岸出入境人员丙型肝炎病毒基因分型研究

目的 了解浙江口岸出入境人员丙型肝炎病毒(HCV)基因型分布特征和病毒变异情况,为丙型肝炎的诊断和预防提供有力依据.方法 荧光定量RT-PCR检测120名出入境人员抗-HCV阳性血清标本,并对其中HCV-RNA阳性的81份血清标本应用基因芯片法进行HCV基因分型,对1b型、2a型、3a型、6型各1株的5 '非编码区(5'NCR)PCR产物测序验证.结果 81份HCv-RNA阳性血清,其中lb型62例,2a型5例,3a型3例,3b型3例,1b/3a型的混合感染2例,1b/3b型3例,6型3例,分别占76.54％、6.17％、2.7％、2.700、2.47％、2.7％、2.7％;1b型的序列与GenBank J-4株的同源性为99.2％,2a型与GenBank J-6株的同源性为99.2％,3a型与GenBank k3a/650株的同源性为97.6％,6型与GenBank Th580株的同源性为96.8％.结论 浙江口岸出入境人员中HCV以1b型为主,3型和2a型次之,少量6型,同时存在HCV1b/3混合型.